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Abnormal assemblies formed by misfolded superoxide dismutase-1 (SOD1) proteins are the likely cause of SOD1-linked familial amyotrophic lateral sclerosis (fALS) and may be involved in some cases of sporadic ALS. To analyze the structure of the insoluble SOD1 amyloid fibrils, we first used limited proteolysis followed by mass spectrometric analysis. Digestion of amyloid fibrils formed from full-length N-acetylated WT SOD1 with trypsin, chymotrypsin, or Pronase revealed that the first 63 residues of the N terminus were protected from protease digestion by fibril formation. Furthermore, every tested ALS-mutant SOD1 protein (G37R, L38V, G41D, G93A, G93S, and D101N) showed a similar protected fragment after trypsin digestion. Our second approach to structural characterization used atomic force microscopy to image the SOD1 fibrils and revealed that WT and mutants showed similar twisted morphologies. WT fibrils had a consistent average helical pitch distance of 62.1 nm. The ALS-mutant SOD1 proteins L38V, G93A, and G93S formed fibrils with helical twist patterns very similar to those of WT, whereas small but significant structural deviations were observed for the mutant proteins G37R, G41D, and D101N. Overall, our studies suggest that human WT SOD1 and ALS-mutants tested have a common intrinsic propensity to fibrillate through the N terminus and that single amino acid substitutions can lead to changes in the helical twist pattern.

As is the case in most mammals, high density lipoproteins (HDL) also comprise the major group of lipid carriers that circulate in bovine (Bos taurus) blood. As a continuation of our proteogenomic studies of mammalian apolipoproteins, we have obtained molecular masses for several of the apolipoproteins associated with bovine HDL. The major apolipoprotein on the HDL surface is apoA-I, but other apolipoproteins were also detected. Using electrospray-ionization mass spectrometry (ESI-MS), we report on values for apolipoproteins, A-I, proA-I and A-II, as well as post-translationally modified apoA-I. Analyses of tryptic fragments did reveal the presence of apoA-IV and apoC-III. However, in contrast to our previous studies of other mammalian HDL, we did not detect apoC-I. Interestingly, examination of the current assembly for the bovine genome does not show any evidence for an apoC-I gene.

The subunit composition of the NAD(P)H dehydrogenase complex of Thermosynechococcus elongatus was analyzed by different types of mass spectrometry. All 15 known subunits (NdhA-NdhO) were identified in the purified NDH-1L complex. Moreover, two additional intact mass tags of 4902.7 and 4710.5 Da could be assigned after re-annotation of the T. elongatus genome. NdhP and NdhQ are predicted to contain a single transmembrane helix each and homologues are apparent in other cyanobacteria. Additionally, ndhP is present in some cyanophages in a cluster of PSI genes and it exhibits partial similarity to NDF6, a subunit of the plant NDH-1 complex.

International audience; A hybrid linear ion-trap Fourier-transform ion cyclotron resonance mass spectrometer was used for top-down characterization of the abundant human salivary Cystatins, including S, S1, S2, SA, SN, C, and D, using collisionally activated dissociation (CAD) after chromatographic purification of the native, disulfide intact proteins from saliva. Post-translational modifications and protein sequence polymorphisms arising from single nucleotide polymorphisms (SNPs) were assigned from precursor and product ion masses at a tolerance of 10 ppm allowing confident identification of individual intact mass tags. Cystatins S, S1, S2, SA and SN were cleaved of a N-terminal 20 amino-acid signal peptide, and Cystatin C a 26-residue peptide, to yield a generally conserved N-terminus. In contrast, Cystatin D isoforms with 24 and 28 amino-acid residue N-terminal truncations were found such that their N-termini were not conserved. Cystatin S1 was phosphorylated at Ser3, while S2 was phosphorylated at Ser1 and Ser3 of the mature protein, in agreement with previous work. Both Cystatin D isoforms carried the polymorphism C46R (SNP: rs1799841). The 14328 Da isoform of Cystatin SN previously assigned with polymorphism P31L due to a SNP (rs2070856) was found only in whole saliva. Parotid secretions contained no detectable Cystatins while whole saliva largely mirrored the contents of submandibular/sublingual (SMSL) secretions. Top-down high-resolution mass spectrometry is a powerful tool for the identification and characterization of potential protein biomarkers in saliva.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Top-down mass spectrometry focuses on intact proteins, thereby avoiding loss of information accompanying 'shotgun' protocols that reduce the proteome to a collection of peptides. A suite of liquid-chromatography technologies has been developed for purification of intact integral membrane proteins in aqueous/organic solvent mixtures compatible with biological 'soft-ionization' mass spectrometry, preserving covalent structure into the gas phase. Multiply charged protein ions are fragmented in the gas phase, using either collision-activated or electron-capture dissociation, thus yielding complex spectra of sequence-dependent product ions that collectively define the original native covalent state of an intact protein. Top down offers a more detail-orientated approach to post-transcriptional and post-translational diversity allowing an enhanced insight beyond genomic translation, which has now extended into the bilayer proteome.

A monoclonal antibody (mAb) has been produced which reacts with human mitofilin, a mitochondrial inner membrane protein. This mAb immunocaptures its target protein in association with six other proteins, metaxins 1 and 2, SAM50, CHCHD3, CHCHD6 and DnaJC11, respectively. The first three are outer membrane proteins, CHCHD3 has been assigned to the matrix space, and the other two proteins have not been described in mitochondria previously. The functional role of this new complex is uncertain. However, a role in protein import related to maintenance of mitochondrial structure is suggested as mitofilin helps regulate mitochondrial morphology and at least four of the associated proteins (metaxins 1 and 2, SAM50 and CHCHD3) have been implicated in protein import, while DnaJC11 is a chaperone-like protein that may have a similar role.

Top-down proteomics characterizes protein primary structures with unprejudiced descriptions of expressed and processed gene products. Gene sequence polymorphisms, protein post-translational modifications, and gene sequence errors can all be identified using top-down proteomics. Saliva offers advantages for proteomic research because of availability and the noninvasiveness of collection and, for these reasons, is being used to search for disease biomarkers. The description of natural protein variants, and intra- and inter-individual polymorphisms, is necessary for a complete description of any proteome, and essential for the discovery of disease biomarkers. Here, we report a striking example of natural protein variants with the discovery by top-down proteomics of two new variants of Peptide P-C. Intact mass measurements, and collisionally activated-, infrared multiphoton-, and electron capture-dissociation, were used for characterization of the form predicted from the gene sequence with an average mass 4371 Da, a form postulated to result from a single nucleotide polymorphism of mass 4372 Da, and another form of mass 4370 Da postulated to arise from a novel protein sequence polymorphism. While the biological significance of such subtle variations in protein structure remains unclear, their importance cannot be assigned without their characterization, as is reported here for one of the major salivary proteins.

Background B. bronchiseptica infections are usually associated with wild or domesticated animals, but infrequently with humans. A recent phylogenetic analysis distinguished two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Complex IV isolates appear to have a bias for infecting humans; however, little is known regarding their epidemiology, virulence properties, or comparative genomics. Results Here we report a characterization of the virulence of human-associated complex IV B. bronchiseptica strains. In in vitro cytotoxicity assays, complex IV strains showed increased cytotoxicity in comparison to a panel of complex I strains. Some complex IV isolates were remarkably cytotoxic, resulting in LDH release levels in A549 cells that were 10- to 20-fold greater than complex I strains. In vivo, a subset of complex IV strains was found to be hypervirulent, with an increased ability to cause lethal pulmonary infections in mice. Hypercytotoxicity in vitro and hypervirulence in vivo were both dependent on the activity of the bsc T3SS and the BteA effector. To clarify differences between lineages, representative complex IV isolates were sequenced and their genomes were compared to complex I isolates. Although our analysis showed there were no genomic sequences that can be considered unique to complex IV strains, there were several loci that were predominantly found in complex IV isolates. Conclusion Our observations reveal a T3SS-dependent hypervirulence phenotype in human-associated complex IV isolates, highlighting the need for further studies on the epidemiology and evolutionary dynamics of this B. bronchiseptica lineage.